Optical microscopes are routinely employed for imaging live cell dynamics. Until recently, conventional optical microscopes lacked the ability to resolve spatial features significantly smaller than the wavelength of light. This kept the structure and dynamics of a vast array of biological processes hidden. Although electron microscopes provide sub-nm spatial resolution images, they are incapable of monitoring live biological dynamics. Understanding the spatial organization and temporal dynamics of nanoscale molecular assemblies is critical to developing a comprehensive understanding of biology. In recent years, sub-diffraction limited microscopes, known as super-resolution (SR) microscopes, have enabled routine live cell imaging at spatial resolutions <50nm. These new tools produced discoveries that challenged multiple paradigms of intracellular processes. Because optical scattering severely distorts SR methods, the SR imaging revolution has failed to be translated deep into scattering tissue. Yet it is well known that the behavior of cells in tissues and tumors deviates strongly from the behavior of 2D cell cultures. Our long-term goal is to develop a new optical SR microscope capable of unlimited super-resolution imaging deep in live animal tissues. This transformational tool will enable discoveries of disease mechanisms that will facilitate the discovery of new treatment paradigms. The objective of this grant is to demonstrate the feasibility of a new approach to optical SR imaging with spatial frequency modulated imaging; this will open the ability for deep-tissue imaging with sub-diffraction limited resolution. The central hypothesis is that spatial frequency modulated SR imaging will exhibit robustness to imaging both for the spatial-temporally modulated illumination light and the collected light used for image formation. Our rationale is that a broad illumination bandwidth will homogenize speckle that would otherwise be accumulated by the spatially structured illumination light. This homogenization prevents the speckle from distorting the image formation process. Scattering of the fluorescent light emitted from the object does not impact the quality of the image. Our specific aims will test the following hypotheses: (Aim1) SR image formation with <50 nm spatial resolution with spatial frequency modulated illumination, and (Aim 2) the suppression of speckle in spatial frequency modulated image formation with broad bandwidth illumination in brain slices. Upon conclusion, we will understand the principles of spatial frequency modulated imaging for imaging deep inside of biological tissues. This contribution is significant since it will establish a new optical microscope technique that will bring the super-resolution imaging revolution deep inside live animal models, opening the path for new discoveries of disease mechanisms. Such discoveries will likely drive an improvement in our understanding of biology and disease, as well as drive the discovery of new diagnostic signatures and treatment targets. The proposed research is innovative because it explores new concepts for imaging with unprecedented spatial resolution at unprecedented depths in optical tissues.